Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:6.3.5.5 (CPS)
 1,262 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme consists of a large synthetase subunit and a small amidotransferase subunit. The small subunit is structurally bilobal. The N-terminal domain is unique compared to the sequences of other known proteins. The C-terminal domain, which contains the direct catalytic residues for the amidotransferase activity of CPS, is homologous to other members of the Triad glutamine amidotransferases. The two domains are linked by a hinge-like loop, which contains a type II beta turn. The role of this loop in the hydrolysis of glutamine and the formation of carbamoyl phosphate was probed by site-directed mutagenesis. Based upon the observed kinetic properties of the mutants, the modifications to the small subunit can be separated into two groups. The first group consists of G152I, G155I, and Delta155. Attempts to disrupt the turn conformation were made by the deletion of Gly-155 or substitution of the two glycine residues with isoleucine. However, these mutations only have minor effects on the kinetic properties of the enzyme. The second group includes L153W, L153G/N154G, and a ternary complex consisting of the intact large subunit plus the separate N- and C-terminal domains of the small subunit. Although the ability to synthesize carbamoyl phosphate is retained in these enzymes, the hydrolysis of glutamine is partially uncoupled from the synthetase reaction. It is concluded that the hinge loop, but not the type-II turn structure of the loop per se, is important for maintaining the proper interface interactions between the two subunits and the catalytic coupling of the partial reactions occurring within the separate subunits of CPS.
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PMID:Role of the hinge loop linking the N- and C-terminal domains of the amidotransferase subunit of carbamoyl phosphate synthetase. 1090 Jan 47

Depending on their physiological role, carbamoyl phosphate synthetases (CPSs) use either glutamine or free ammonia as the nitrogen donor for carbamoyl phosphate synthesis. Sequence analysis of known CPSs indicates that, regardless of whether they are ammonia- or glutamine-specific, all CPSs contain the structural equivalent of a triad-type glutamine amidotransferase (GAT) domain. In ammonia-specific CPSs, such as those of rat or human, the catalytic inactivity of the GAT domain can be rationalized by the substitution of the Triad cysteine residue by serine (1). The ammonia-specific CPS of Rana catesbeiana (fCPS) presents an interesting anomaly in that, despite its retention of the entire catalytic triad (2) and almost all other residues conserved in Triad GATs, it is unable to utilize glutamine as a nitrogen-donating substrate (3). Based on our earlier work with the glutamine-utilizing E. coli CPS (eCPS), we have targeted residues Lys258 and Glu261 in the fCPS GAT domain as critical for preventing GAT function. Previously we have shown that substitution of the corresponding residues in eCPS by their fCPS counterparts (Leu --> Lys and Gln --> Glu) resulted in complete loss of GAT function in eCPS (3). To examine the role of these residues in the fCPS GAT component, we have cloned the full-length fCPS gene from R. catesbeiana liver. Here we report the first heterologous expression of an ammonia-specific CPS and show that a single mutation of the frog enzyme, K258L, yields a gain of glutaminase function.
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PMID:Gain of glutaminase function in mutants of the ammonia-specific frog carbamoyl phosphate synthetase. 1273 80